Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.     

Growth Conditions Differentially Regulate the Expression of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate (AMPA) Receptor Subunits in Cultured Neurons

We have studied the expression of a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K^± (10, 25, or 40 mM) with or without 100 μM N-methyl-D-aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K^± (K10) and increased dramatically in cultures grown in 25 mM K^± (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 ± NMDA). In cultures grown in 40 mM K^± (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and -3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA-stimulated ^4SCa^2± influx and 40-[³H]phorbol 12, 13-dibutyrate binding in intact cells. Both functional parameters increased along with the K^± concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits. These results indicate that functional diversity of AMPA receptors can be generated by the degree of chronic depolarization and/or exposure to NMDA in neurons developing in primary culture.     

Thyroidal regulation of different isoforms of NaKATPase in the primary cultures of neurons derived from fetal rat brain

The developmental profile of the different isoforms of NaKATPase have been investigated using primary cultures of isolated neurons initiated from 17 day old fetal rat brain. Northern blot analysis showed that the expression of three alpha isoforms (alpha(1), alpha(2) and alpha(3)) and two beta isoforms (beta(1) and beta(2)) increased progressively and reached a peak between 12 to 16 days of culture. Comparison of the mRNA levels of these isoforms in the cells maintained in thyroid hormone deficient (TH def) and thyroid hormone supplemented (TH sup) media for 6-12 days, revealed for the first time that in the neurons three alpha and two beta isoforms of NaKATPase are sensitive to TH. Furthermore immunocytochemical staining of these cells with isoform specific NaKATPase antibodies showed that the uniform distribution of alpha(2), alpha(3) and beta(2) isoforms in the neuronal processes require the presence of TH. These results establish neurons as the target cells for the regulation of NaKATPase by TH in the developing brain.     

Tumor necrosis factor p55 and p75 receptors are involved in chemical-induced apoptosis of dentate granule neurons

Localized tumor necrosis factor-alpha (TNFalpha) elevation has diverse effects in brain injury often attributed to signaling via TNFp55 or TNFp75 receptors. Both dentate granule cells and CA pyramidal cells express TNF receptors (TNFR) at low levels in a punctate pattern. Using a model to induce selective death of dentate granule cells (trimethyltin; 2 mg/kg, i.p.), neuronal apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ end labeling, active caspase 3 (AC3)] was accompanied by amoeboid microglia and elevated TNFalpha mRNA levels. TNFp55R (55 kDa type-1 TNFR) and TNFp75R (75 kDa type-2 TNFR) immunoreactivity in AC3(+) neurons displayed a pattern suggestive of receptor internalization and a temporal sequence of expression of TNFp55R followed by TNFp75R associated with the progression of apoptosis. A distinct ramified microglia response occurred around CA1 neurons and healthy dentate neurons that displayed an increase in the normal punctate pattern of TNFRs. Neuronal damage was decreased with i.c.v. injection of TNFalpha antibody and in TNFp55R-/-p75R-/- mice that showed higher constitutive mRNA levels for interleukin (IL-1alpha), macrophage inflammatory protein 1-alpha (MIP-1alpha), TNFalpha, transforming growth factor beta1, Fas, and TNFRSF6-assoicated via death domain (FADD). TNFp75R-/- mice showed exacerbated injury and elevated mRNA levels for IL-1alpha, MIP-1alpha, and TNFalpha. In TNFp55R-/- mice, constitutive mRNA levels for TNFalpha, IL-6, caspase 8, FADD, and Fas-associated phosphatase were higher; IL-1alpha, MIP-1alpha, and transforming growth factor beta1 lower. The mice displayed exacerbated neuronal death, delayed microglia response, increased FADD and TNFp75R mRNA levels, and co-expression of TNFp75R in AC3(+) neurons. The data demonstrate TNFR-mediated apoptotic death of dentate granule neurons utilizing both TNFRs and suggest a TNFp75R-mediated apoptosis in the absence of normal TNFp55R activity.     

GABAA alpha6-containing receptors are selectively compromised in cerebellar granule cells of the ataxic mouse, stargazer

Stargazer mice fail to express the gamma2 isoform of transmembrane alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 +/- 6% fewer inhibitory gamma-aminobutyric acid, type A (GABA(A)), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABA(A) receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (alpha6betaxdelta) alpha6-containing GABA(A) receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABA(A) receptor alpha6, delta (mature cerebellar granule cell-specific proteins), and beta3 subunit expression in stargazer. The subunit composition of the residual alpha6-containing GABA(A) receptors was unaffected by the stargazer mutation. However, we did find evidence of an approximately 4-fold up-regulation of alpha1betadelta receptors that may compensate for the loss of alpha6-containing GABA(A) receptors. PCR analysis identified a dramatic reduction in the steady-state level of alpha6 mRNA, compatible with alpha6 being the primary target of the stargazer mutation-mediated GABA(A) receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic alpha6-containing GABA(A) receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.     

Neuregulin induces GABAA receptor beta2 subunit expression in cultured rat cerebellar granule neurons by activating multiple signaling pathways

The GABAA receptor beta subunit is required to confer sensitivity to gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the CNS. In previous studies we demonstrated that the growth and differentiation factor neuregulin 1 (NRG1) selectively induced expression of the beta2 subunit mRNA and encoded protein in rat cerebellar granule neurons in culture. In the present report we examine the signaling pathways that mediate this effect. These studies demonstrate that the effects of NRG1 on beta2 subunit polypeptide expression require activation of the ErbB4 receptor tyrosine kinase; its effects are inhibited by pharmacological blockade of ErbB4 phosphorylation or reduction of receptor level with an antisense oligodeoxynucleotide. The NRG1-induced activation of ErbB4 stimulates the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and cyclin-dependent kinase-5 (cdk5) pathways. Pharmacological blockade of any of these pathways inhibits increased beta2 subunit expression, demonstrating that all three pathways are required to mediate the effects of NRG1 on GABAA receptor subunit expression in cerebellar granule neurons. These studies provide novel information concerning the actions of NRG1 on GABAA receptor expression in the CNS.     

Endogenous Amino Acid Release from Cultured Cerebellar Neuronal Cells: Effect of Tetanus Toxin on Glutamate Release

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.     

An early increase in the disialoganglioside GD3 contributes to the development of neuronal apoptosis in culture

We induced apoptosis in primary cultures of cerebellar granule neurons by switching the growing medium into a medium containing lower concentrations of K(+) (5 or 10 mM instead of 25 mM) or, alternatively, by addition of staurosporine. The apoptotic phenotype was always preceded by an early increase in the intracellular levels of the disialoganglioside GD3, which peaked at 2-6 h and returned back to normal at 12 h. GD3 synthase, the enzyme that forms GD3 from the monosialoganglioside GM3, was also induced at early times after the induction of apoptosis in granule cells. Immunofluorescent staining showed that GD3 increased in neuronal cell bodies and neurites, but was never localized in cell nuclei. In cultures switched into a low K(+)-containing medium, exogenously applied GD3, but not the disialoganglioside GD1a, accelerated the development of neuronal apoptosis. In contrast, the antisense-induced knock-down of GD3 synthase was protective against granule cell death induced by lowering extracellular K(+) from 25 to 10 - but not 5 - mM. These results demonstrate that an early and transient increase in GD3 synthesis is one of the factors that contribute to the induction of neuronal apoptosis in culture.     

Up-Regulation of NR2B Subunit of NMDA Receptors in Cerebellar Granule Neurons by Ca2+/Calmodulin Kinase Inhibitor KN93

Abstract: Recordings of NMDA-activated currents from cerebellar granule neurons in culture revealed a developmental increase in current density accompanied by a slight decrease of the half-maximal effective concentration. At the same time, a decrease of NMDA receptors comprising NR2B subunits was demonstrated by the reduction in the antagonism of NMDA currents by ifenprodil. Ifenprodil antagonism increased after treatment for 24 h with KN93- and KN62-selective inhibitors of the Ca²⁺/calmodulin-dependent protein kinases (CaM kinases), indicating a selective increase of receptor containing NR2B subunit. This increase was observed at all ages tested: 4 days in vitro (DIV4), DIV6, and DIV13. Western blot analysis with specific NMDA receptor antibodies performed at DIV6 confirmed the electrophysiological data. At this age, the negative control KN92 was ineffective. The increasing ifenprodil antagonism after KN93 treatment was proportionally greater in cells at DIV13 than at DIV4. Treatment with NMDA (100 µ M ) of cerebellar cultures for 24 h produced a decrease in the NMDA-induced current density by almost 50% at all ages tested. Ifenprodil antagonism, however, was unchanged. We propose that the expression of NR2B subunits in cerebellar granule cells is selectively stimulated by the inhibition of CaM kinases.     

Developmental expression of N-methyl-d-aspartate (NMDA)-induced neurotoxicity,NMDA receptor function,and the NMDAR1 and glutamate-binding protein subunits in cerebellar granule cells in primary cultures

Cerebellar granule cells maintained in vitro as primary cultures are a relatively homogeneous neuronal population that can be used to evaluate the developmental expression of neurotransmitter receptors and to assess their role in cell survival and degeneration. The toxicity induced by N-methyl-d-aspartate (NMDA) in granule cells maintained under partially depolarizing conditions and in the presence of physiologic extracellular concentrations of Mg²⁺ was greatest for the neurons maintained for 14 days in vitro (DIV). However, following NMDA receptor activation neurons as young as 5 DIV exhibited increases in the concentration of intracellular free Ca²⁺ which were as large as those achieved with cells at 8–9 or 13–14 DIV. The less mature neurons exhibited a down-regulation of responses to increasing concentrations of NMDA and the more mature cells maintained elevated intracellular Ca²⁺ levels during the inter-stimulus periods. Immunochemical analyses of the expression of the NMDA receptor-associated proteins NMDAR1 and glutamatebinding protein (GBP) in granule cells indicated a developmental increase in both proteins, albeit the pattern of expression of NMDAR1 was the more complex. No definite correlation has yet been established between toxicity induced by NMDA and the expression of these two proteins. Finally, although the developmental expression of nitric oxide synthase, an enzyme that catalyzes the formation of the potentially neurotoxic radicals nitric oxide and superoxide anion, increased progressively with the maturation of neurons in culture, an inhibitor of this enzyme did not protect neurons from NMDA-induced toxicity. Therefore, the developmental changes in granule cells that lead to increased vulnerability following excessive activation of NMDA receptors are not yet completely defined.Special issue dedicated to Dr. Robert Balázs     

gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA(A) receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABA(A) receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABA(A) receptor subunit mRNA expression, including greater than two-fold lower expression of alpha1-, alpha4- and gamma2-subunit mRNAs and 10-fold higher expression of alpha5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA(A) receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABA(A) receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABA(A) receptor function during postnatal development.     

Long-Term GABA Treatment Elicits Supersensitivity of Quisqualate-Preferring Metabotropic Glutamate Receptor in Cultured Rat Cerebellar Neurons

Abstract: In primary cultures of rat cerebellar granule neurons, GABA treatment (50 μ M , 7 days) caused a withdrawal supersensitivity selective for the metabotropic glutamate receptors that mainly prefer l -glutamate, quisqua- late and, to a lesser extent, kainate. The withdrawal supersensitivity was absent when 10 μ M SR-95531 was coadministered with GABA during the treatment period, an event that suggests the GABA_A receptors primarily produced the GABA treatment effect. This was supported further by the inability of baclofen treatment to mimic completely the treatment effect of GABA. Withdrawal from 7 days of baclofen treatment only produced a slight increase in the metabotropic effect of l -glutamate and carbachol. In addition, in untreated neurons, baclofen had no acute effect, whereas GABA inhibited the effect of l -glutamate and carbachol. The inhibitory effect of GABA was reversed by SR-95531 and was absent in neurons treated with GABA. These observations suggest the involvement of GABA_A receptors and the apparent development of tolerance to GABA, respectively. Also, dependence on GABA may have occurred; the metabotropic effects of glutamate, kainate, and quisqualate were not altered in neurons maintained with GABA treatment.     

Expression of Neurotransmitter Receptors by mRNAs from Neurons Developing In Vitro: A Xenopus Oocyte Expression Study

Abstract: Poly(A)⁺ mRNA was extracted from cultures of neurons isolated from mouse embryonic day 14 cerebral cortex and injected into Xenopus oocytes. This led to the expression of receptors for γ-aminobutyric acid (GABA), glycine, acetylcholine, serotonin, glutamate, kainate, N -methyl-D-aspartate, and quisqualate. Northern blot analysis of poly(A)⁺ mRNA from the cultured neurons with a GluRI cDNA probe revealed the presence of three hybridization bands with estimated mRNA sizes of 5.1, 4.0, and 3.1 kb, respectively. The development of mRNAs coding for neurotransmitter receptors was investigated by isolating mRNA from neurons cultured for 2, 8, and 14 days in vitro and injecting it into Xenopus oocytes. The amplitude of membrane currents elicited by the transmitters gave a measure of the relative amounts of the different mRNAs. The size of the responses to kainate, aspartate (together with glycine), glutamate, acetylcholine, GABA, serotonin, and glycine increased with the time of culture in vitro. However, in contrast to all other agonist-induced currents, the current induced by glycine failed to increase further from 8 to 14 days in culture. It is concluded that the time course of receptor development in cortical neurons in vitro is similar to the development in vivo.     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.     

Functional GABA(B) receptors expressed in cultured calvarial osteoblasts

In immature and mature primary cultured rat calvarial osteoblasts, both mRNA and corresponding proteins were constitutively expressed for 2 splice variants of GABA(B) receptor (GABA(B)R) subunits but not for any known GABA(A) and GABA(C) receptor subunits. The agonist for GABA(B)R baclofen significantly inhibited cAMP formation induced by forskolin in a manner sensitive to the antagonist 2-hydroxysaclofen. Similar expression was seen with mRNA for GABA(B)R-1a and -1b splice variants in the murine calvarial osteoblast cell line MC3TC-E1 cells cultured for 7-21 days in vitro (DIV). In these MC3T3-E1 cells, baclofen not only inhibited the activity of alkaline phosphatase, but also exacerbated Ca2+ accumulation, throughout the culture period up to 28 DIV. These results suggest that GABA may play an unidentified role in mechanisms associated with cellular proliferation, differentiation, and/or development through functional GABA(B)R constitutively expressed in cultured osteoblasts.     

Metabotropic Glutamate Receptors in Cultured Cerebellar Granule Cells: Developmental Profile

Abstract: Excitatory amino acid (EAA)-induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR) agonists, including L-Glu, quisqualate, and trans -(±)-1-amino-1,3-cyclopentanedicarboxylic acid: The stimulation of [³H]inositol monophosphate ([³H]-InsP) formation was low at 2 days in vitro (DIV), but the response increased steeply, reaching a peak at 4 DIV, followed by a progressive decline. In contrast, carbamylcholine-induced PPI hydrolysis exhibited a plateau after a pronounced increase during the first week in vitro. At 6 DIV, but not at 4 DIV, when the activity peaked, PPI hydrolysis elicited by Glu was reduced by the N -methyl- d -aspartate (NMDA) receptor antagonist MK-801, indicating that in cultured granule cells, NMDA receptors contribute to [³H]-InsP formation and that this component of the response develops relatively late. Accordingly, NMDA-induced [³H]-InsP formation, estimated under Mg²⁺-free conditions, increased markedly from very low values at 2 DIV to a plateau at 8–10 DIV. The developmental pattern of EAA-induced PPI hydrolysis was paralleled by changes in the level of an mRNA for a specific mGluR subtype ( mGluR1 mRNA). RNA blot analysis performed with the pmGR1 cDNA probe revealed that the hybridization signal in RNA extracts from cultures at 1 DIV was very weak, but mGluR mRNA levels increased dramatically between 1 and 3 DIV, followed by a progressive decrease, so that by 15 DIV the mRNA levels were only ∼10% of the values at 3 DIV. These observations indicate that the functional expression of the mGluR is subject to developmental regulation, which critically involves receptor mRNA levels.